541–551 QUARTERLY Review Mitochondrial potassium and chloride channels ��

Channels selective for potassium or chloride ions are present in inner mitochondrial membranes. They probably play an important role in mitochondrial events such as the formation of delta pH and regulation of mitochondrial volume changes. Mitochondrial potassium and chloride channels could also be the targets for pharmacologically active compounds such as potassium channel openers and antidiabetic sulfonylureas. This review describes the properties, pharmacology, and current observations concerning the functional role of mitochondrial potassium and chloride channels.


MITOCHONDRIAL ATP-REGULATED POTASSIUM CHANNEL
In 1991, the mitochondrial ATP-regulated potassium channel (mitoK ATP channel) was identified by patch-clamp single-channel recordings in the inner membrane of rat liver mitochondria (Inoue et al., 1991).Its activity was later reconstituted into liposomes containing partially purified proteins from the inner membrane of beef heart mitochondria (Paucek et al., 1992).This channel is blocked not only by ATP but also by antidiabetic sulfonylureas and 5-hydroxydecanoic acid (5-HD) (Jaburek et al., 1998) and is activated by some potassium channel openers (KCOs) such as diazoxide (Garlid et al., 1996).According to the present knowledge, the mitoK ATP channel may have a dual physiological function (Szewczyk, 1998).First, it can maintain potassium homeostasis within mitochondria and thus control mitochondrial volume (Halestrap, 1994).Second, potassium uptake upon mitochondrial energization may partly compensate the electric charge transfer produced by proton pumping and thus enable the formation of a pH gradient along with transmembrane electric potential (Czy¿ et al., 1995).
The molecular identity of mitoK ATP channels is unknown (for review, see Szewczyk & Marban, 1999).Several observations on the pharmacological profile and immunofluorescence may suggest that the mitoK ATP channel belongs to the inward rectifier K + channel family (Suzuki et al., 1997).Using immunofluorescence and immunogoldstaining, it has been shown that the inward rectifier K + channel, Kir6.1, is present in rat skeletal muscle and liver mitochondria (Suzuki et al., 1997).Recently, Kir6.1 was also localised in rat brain by in situ hybridization and immunohistochemistry (Zhou et al., 1999).The mRNA of Kir6.1 was ubiquitously expressed in various neurons and glial cells.Interestingly, under electron microscope, the immunoreactive products were specifically restricted to the mitochondria (Zhou et al., 1999).Similar ATP-regulated K + channels are present in the plasma membrane of pancreatic B-cells, smooth, skeletal and cardiac muscle cells and of neurons (Lazdunski, 1994).The pore subunit of these channels, Kir6.1 or Kir6.2, together with the antidiabetic sulfonylureas receptor (SUR), constitutes the functional plasma membrane ATP-regulated K + channel (K ATP channel) (Aguilar- Bryan et al., 1995;Inagaki et al., 1995).Plasma membrane K ATP channels are specifically activated by drugs known as potassium channel openers (KCOs) (Edwards & Weston, 1993).Garlid et al. (1996) demonstrated that the heart and liver mitoK ATP channel share some pharmacological properties with the plasma membrane K ATP channel, while possessing a distinct pharmacological profile.The outstanding pharmacological signature of mitoK ATP channels is their sensitivity to the opening by diazoxide, exceeding the sensitivity of cardiac plasma membrane K ATP channels by about 1 000-fold.This observation was crucial for further establishing the functional role of mitoK ATP channels in cardiomyocytes.
Studies on the mitoK ATP channel can be divided into two periods.The first, starting in 1991, could be named "The mitoK ATP channel as an interesting object".This first part was dedicated to the biochemical characterisation of the mitoK ATP channel and searching for its physiological function in cellular bioenergetics.The second period, starting in 1997, could be named "The mitoK ATP channel as an important cellular effector".This part concerns studies providing evidence for the involvement of the mitoK ATP channel in cardioprotection.Both periods are linked by the discovery of Garlid's group that the potassium channel opener, diazoxide, and potassium channel inhibitor, 5-HD, act potently on the heart mitoK ATP channel but not on the heart plasma membrane K ATP channel.Since this observation, a variety of reports have been published suggesting that the mitoK ATP channel plays an important role in cardioprotection.Here, the current knowledge about the mitoK ATP channel will be briefly reported.

MITOCHONDRIAL ATP-REGULATED POTASSIUM CHANNEL IN CARDIO-PROTECTION
Applying KCOs, such as diazoxide, and the mitoK ATP channel inhibitor 5-HD, it was shown that the mitoK ATP channel could be an effector of ischemic preconditioning in heart (for review see Gross & Fryer, 1999).
Lethal ischemic injury to the heart can be dramatically blunted by brief periods of ischemia known as "ischemic preconditioning".Despite intensive investigation, the molecular mechanism of preconditioning remains poorly understood.Nevertheless, K ATP channels are clearly involved: KCOs mimic preconditioning in the absence of ischemia, while K ATP channel blockers such as glibenclamide and 5-HD abolish the beneficial effects of preconditioning ischemia.The initial hypothesis to explain these observations involved plasma membrane K ATP channels.Recently, in order to compare cardioprotective potency, diazoxide and cromakalim were given to isolated rat hearts subjected to global ischemia and reperfusion (Garlid et al., 1997).Diazoxide and cromakalim increased the time of onset of contracture with similar potency and improved postischemic functional recovery in a glibenclamide-sensitive manner.In addition, 5-HD completely abolished the protective effect of diazoxide.A conclusion of this study was that diazoxide protected ischemic hearts by opening mitoK ATP channels rather than plasma membrane K ATP channels.In a similar approach, Liu et al.
(1998) indexed mitoK ATP and surface K ATP channel activity simultaneously in cardiac myocytes.This approach enabled the function of mitoK ATP channels to be assayed in intact cells.MitoK ATP channel activity was indexed by measuring flavoprotein fluorescence, an endogenous reporter of the mitochondrial re-dox state.Opening of mitoK ATP channels dissipates the mitochondrial membrane potential established by the respiratory chain.This dissipation accelerates electron transfer by the respiratory chain, and leads to net oxidation of mitochondria that can be monitored by recording the fluorescence of FAD-linked enzymes in mitochondria.Low concentrations of the KCO diazoxide have been reported to activate mitoK ATP channels while cardiac plasma membrane K ATP channels are quite resistant to this drug.It was shown that diazoxide induced reversible oxidation of the mitochondrial flavoproteins but did not activate plasma membrane K ATP channels.The subcellular site of diazoxide action was localized to mitochondria by confocal imaging of flavoprotein fluorescence and mitochondria stained with fluorescent probe tetramethylrhodamine ethyl ester (TMRE).It was also shown that the flavoprotein fluorescence affected by diazoxide colocalizes precisely with the staining pattern for TMRE in the same cardiomyocyte.These studies also established that 5-HD is a specific blocker of the mitoK ATP channel, at least in heart muscle cells.In a cellular model of cardiomyocyte ischemia, diazoxide prevented cell death to the same degree as preconditioning -an effect which was blocked by 5-HD (Liu et al., 1998).
Initial observations of the cardioprotective action of diazoxide were further confirmed and developed by a series of reports: u Isolated mitochondria from rat heart were used to examine the effect of KCOs on mitochondrial membrane potential, respiration, ATP generation, Ca 2+ transport and matrix volume (Holmuhamedov et al., 1998) Liang & Gross, 1999).These reports suggested that stimulation of the d 1 -opioid receptor before ischemic insult produces a delayed cardioprotective effect that is possibly the result of mitoK ATP channel activation.u Recently, it has been shown that ischemic preconditioning depends on the interaction between the actin cytoskeleton and mitoK ATP channel (Baines et al., 1999b).
For the evaluation of the participation of these proposed end effectors, rabbit hearts underwent regional ischemia and reperfusion.It was shown that diazoxide ad-ministered before ischemia was protective.Similarly, anisomycin, a p38/JNK activator, reduced infarct size but protection from both diazoxide and anisomycin was abolished by 5-HD, an inhibitor of the mitoK ATP channel.Interestingly, the protection by preconditioning, diazoxide, or pinacidil could be abolished by disruption of the cytoskeleton by cytochalasin D. These data suggest that both the mitoK ATP channel and the cytoskeletal protein actin are important in protecting hearts by preconditioning.u Ischemic tolerance of the heart can also be increased by long-term exposure of animals to chronic hypoxia associated with natural or stimulated high altitude.Recently, it has been concluded that long-term adaptation of rats to high altitude hypoxia decreases the susceptibility of their hearts to ischemic arrythmias (Asemu et al., 1999).The mitoK ATP channel, rather than the plasma K ATP channel, appeared to be involved in this protective mechanism (Asemu et al., 1999).u The role of PKC in the mitoK ATP channel-mediated protection against Ca 2+ overload injury of the rat myocardium was shown (Wang & Ashraf, 1999).Massive cell damage occurs in the heart within seconds when it is perfused with a medium devoid of Ca 2+ followed by perfusion with a solution that contains Ca 2+ .This phenomenon has been called the Ca 2+ paradox (Ca 2+ PD).A mild stress induced by brief Ca 2+ depletion and repletion, called Ca 2+ preconditioning, has been shown to protect the myocardium from Ca 2+ PD injury or subsequent sustained ischemia and reperfusion.It has been tested if the protection by diazoxide reduces the Ca 2+ paradox, whether diazoxide mimics the effects of Ca 2+ preconditioning, and whether diazoxide reduces Ca 2+ paradox injury via the PKC signalling pathway.
The salutary effects of diazoxide on Ca 2+ PD injury were similar to those in hearts that underwent Ca 2+ preconditioning or pretreatment with PMA before Ca 2+ PD.The addition of 5-HD or chelerythrine during diazoxide pretreatment completely abolished the beneficial effects of diazoxide.PKC-d was translocated to the mitochondria, intercalated disks and nuclei of myocytes in diazoxide-pretreated hearts, and PKC-a and PKC-e were translocated to the sarcolemma and intercalated disks, respectively.This study suggested that the mitoK ATP channel activity is mediated by the PKC signalling pathway.u It has been observed that plasma membrane K ATP channel inhibitors administered before ischemic preconditioning did not significantly attenuate cardioprotection (Fryer et al., 2000).On the contrary, pretreatment with 5-HD before ischemic preconditioning partially abolished cardioprotection.Moreover, it has been shown that rats subjected to ischemia-reperfusion synthesise much less ATP than control animals and ischemic preconditioning significantly increases ATP synthesis when 5-HD is administered before ischemic preconditioning (Fryer et al., 2000).These data are consistent with the notion that inhibition of mitoK ATP channels attenuate ischemic preconditioning by reducing ischemic preconditioning-induced protection of mitochondrial function.u Evidence for mitoK ATP channels as effectors of human myocardial preconditioning was shown (Ghosh et al., 2000).u Preservation of mitochondrial function by diazoxide during sustained ischemia in rat heart was observed (Iwai et al., 2000).It has been shown that hypoxia induces a decrease in the mitochondrial oxygen consumption rate of myocardial skinned bundles to approximately 40% of the pre-hypoxic value.In contrast, treatment of the bundles with diazoxide preserves the normal mitochondrial oxygen consumption rate during hypoxia.This effect was abolished by the combined treatment with either glibenclamide or 5-HD (Iwai et al., 2000).
u Activation of the mitoK ATP channel by nitric oxide was shown (Sasaki et al., 2000).
The NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) oxidised the mitochondrial matrix dose-dependently without activating the plasma membrane K ATP channel.SNAP-induced oxidation was blocked by 5-HD and by a NO scavenger.The conclusion of this study was that NO directly activates mitoK ATP channels and potentates the ability of diazoxide to open these channels.It has been shown that diazoxide induces both early and delayed anti-ischemic effects via the opening of mitoK ATP channels, which is nitric oxide-dependent (Ockaili et al., 1999).Very recently, the effects of KCOs on intracellular Ca 2+ concentration and mitochondrial potential (DY m ) in cultured rat hippocampal neurons have been studied (Jakob et al., 2000).Using Western blots and immunochemistry it was shown that pretreatment with the KCOs cromakalim and diazoxide increased the expression level of proteins involved in apoptosis.These results also suggest that mitoK ATP channels are present in hippocampal neurons and may confer neuroprotection by altering Bcl2 and Bcl-XL expression levels.

MITOCHONDRIAL LARGE CONDUC-TANCE POTASSIUM CHANNEL
In 1999, a large conductance BK-type potassium channel was identified by patch-clamp techniques in the inner mitochondrial membrane of the human glioma cell line LN229 (Siemen et al., 1999).This channel shows similarities to the BK-type K (Ca) channel of the plasma membrane found in several tissues (Latorre et al., 1989) and in cell granules of giant green alga Chara australis (Laver & Walker, 1991).
Conductance of the mitochondrial BK-type K (Ca) channel is 295 pS and it shows a linear dependence of single channel current from 546 A. Kiciñska and others 2000 voltage in 150 mM KCl solutions on either side of the inner mitochondrial membrane.This channel is activated by Ca 2+ (EC 50 = 0.9 mM at 60 mV) and the open probability increases with increasing Ca 2+ concentration.It is unknown whether there is a Ca 2+ -binding site on the matrix or cytosolic side of the mitochondrial membrane (Siemen et al., 1999).Like most BK-type channels, the mitochondrial channel is blocked by charybdotoxin in a dose dependent manner (EC 50 = 1.4 nM) (Siemen et al., 1999).
The function of the mitochondrial BK-type K (Ca) channel is unknown.In the plasma membrane it is thought to link the membrane potential to cellular metabolism (Petersen, 1992).It plays an important role in secretion and in repolarizing phase of action potential in some neurons and myotubules (Latorre et al., 1989).It has been speculated that the BK-channel in the human glioma cell LN229 can cause the complete and irreversible uncoupling of mitochondria, thereby promoting apoptosis (Siemen et al., 1999).

MITOCHONDRIAL CHLORIDE CHANNEL
Chloride channels are involved in several crucial cell processes regulating cell volume, membrane potential, transepithelial transport, signal transduction and acidification of organelles (Jentsch & Gunther, 1997).Recently, a novel intracellular chloride channel (mtCLIC) has been identified and shown to be localised in mitochondria (Fernández-Salas et al., 1999).The mtCLIC cDNA is very similar to several reported sequences in the GeneBankÔ/EMBL/Protein Data Bank database.It is similar to several plasma membrane chloride channels and intracellular chloride channels (93% identity with rat intracellular chloride channel p64H1, 88% homology with human H1 chloride channel and 87% homology with human intracellular chloride channel p64H1) (Fernández-Salas et al., 1999).The mtCLIC cDNA codes for a 253 amino-acid protein with a predicted molecular mass of 27.8 kDa (Fernández-Salas et al., 1999).mtCLIC protein also shows extensive homology with a family of chloride channels, especially the intracellular p64H1 chloride channel present in the endoplasmic reticulum of rat brain (98% homology) (Duncan et al.,1997) and human p64H1 (Chuang et al., 1999).The analysis of mtCLIC protein revealed two putative transmembrane domains and possible cAMP-dependent protein kinase phosphorylation sites, several PKC, CK2 and tyrosine kinase phosphorylation sites and N-myristoylation sites (Fernández-Salas et al., 1999).mtCLIC mRNA is expressed to a greatest extent in vivo in heart, lung, liver, brain and skin.However, it has been detected in all tested tissues (Fernández-Salas et al., 1999).This protein is the first intracellular ion channel shown to be differentially regulated.The expression in heart, lung, liver, kidney and skin is higher in p53 +/+ mice than in p53 -/- mice ranging from a 2.5-to 5-fold difference but these differences were not detected in intestine, spleen, testis and kidney (Fernández-Salas et al., 1999).Also, in cultured keratinocytes the levels of mtCLIC mRNA and protein are higher in p53 +/+ keratinocytes than in p53 -/-cells and further increased after induction of differentiation of keratinocytes (Fernández-Salas et al., 1999).Moreover, overexpression of p53 in primary mouse keratinocytes increases mtCLIC mRNA and the protein level.Exogenous human TNFa also increase the levels of mtCLIC mRNA and protein in both p53 +/+ and p53 -/-keratinocytes (Fernández-Salas et al., 1999).mtCLIC is the first chloride channel shown to be localised in mitochondria.It has been shown that other proteins belonging to the CLIC family of intracellular chloride channels are involved in the control of transmembrane potential, ionic concentration and pH in intracellular organelles such as early endosomes (Edwards et al., 1998).The mito-chondrial chloride channel may be very important for establishing the pH gradient across the inner mitochondrial membrane, as chloride currents could compensate for charges during H + transport through the respiratory chain.
As mentioned above, mtCLIC expression is regulated by p53 and TNFa, proteins that are both important in apoptotic cell death (Fernández-Salas et al., 1999).It has been suggested that mtCLIC can provide a potential common downstream effector for the p53 and TNFa pathways (Fernández-Salas et al., 1999).This is an interesting hypothesis as both pathways can mediate UV-induced apoptosis in keratinocytes (Schwarz et al., 1995;Ziegler et al., 1994).It is possible that changes in inner mitochondrial membrane permeability to chloride ions and changes in mitochondrial membrane potential lead to the activation of mitochondrial permeability transition and apoptosis.

FINAL REMARKS AND FUTURE PERSPECTIVES
Due to experimental difficulties, mitochondrial K + and Cl -channels are challenging targets for basic research.Recent observations on the role of mitoK ATP channels in ischemic preconditioning open new perspectives for this field.For example, ischemic preconditioning has recently emerged as a new strategy for improving the preservation of heart transplants.Hence, there is a continuing effort to identify endogenous mediators of the preconditioning-induced signalling pathway in an attempt to use some of them therapeutically.Recently, using a model of prolonged cold heart storage, it has been shown that the mitoK ATP channel opener diazoxide can reproduce the protection conferred by ischemic preconditioning (Kevelaitis et al., 1999).Both ischemic and diazoxide preconditioning provided a similar degree of cardioprotection.These beneficial effects were abolished by 5-HD pretreatment.These data support the concept that the cardioprotective effects of ischemic preconditioning can be simulated by a mitoK ATP channel opener and suggest that the activation of these channels could be an effective means of improving the preservation of globally ischemic cold-stored hearts, as occurs during cardiac transplantation (Ahmet et al., 2000;Kevelaitis et al., 1999).This example illustrates that the understanding of mitochondrial ion channel function could not only significantly deepen our knowledge of cellular physiology but also could lead to the better treatment of cardiovascular diseases.